Nitric oxide (NO) is a signaling molecule synthesized from l-arginine by nitric oxide synthases (NOSs). NOS isoforms are either constitutive (endothelial NOS [eNOS] and neuronal NOS [nNOS]) or inducible NOS (iNOS). Previously, our group has reported that NO is expressed during and modulates fracture healing. In this study, we evaluated the specific contribution of iNOS to fracture healing by using iNOS gene therapy in iNOS-deficient mice. Twelve-week-old female wild-type mice and iNOS-KO mice had a right femoral midshaft osteotomy fixed with an intramedullary 0.5-mm-diameter needle. A gelatine sponge was implanted across the fracture site. The gelatine sponge received either Ad5-CMViNOS (in iNOS-deficient mice; n = 16) or Ad5-CMVempty (in wild-type mice; n = 15, and iNOS-deficient mice; n = 15) at a dose of 107 pfu. Mice were sacrificed at day 14, and their right and left hind limbs were harvested. Cross-sectional area (CSA) was determined by measuring the callus diameter across the mediolateral and anteroposterior plane using a vernier caliper. Specimens were loaded to failure torsionally in a biaxial INSTRON testing system, and maximum torque, torsional stiffness, and maximal and total energy were determined.Deletion of the iNOS gene decreased the total and maximum energy absorption of the healing femoral fracture by 30% and by 70% (P P = 0.01) in comparison to iNOS(-/-) mice that did not receive the iNOScDNA. There were no significant differences in the biomechanical properties of intact femora.These data indicate that iNOS is important in mouse fracture healing. However, the clinical utility of NOS gene therapy to enhance fracture healing will need further evaluation.